Cdc7-ask kinase complex, substrates of the kinase complex, antibody specific to the substrate, and method of screening compound capable of inhibiting cdc7-ask kinase using the same

ABSTRACT

The present invention provides methods for measuring the phosphorylation activity of Cdc7-ASK kinase complex by using as an indicator the level of phosphorylation at a phosphorylation site of MCM, which is a substrate of Cdc7-ASK kinase complex. The effects of test compounds on the phosphorylation activity of Cdc7-ASK kinase complex can also be evaluated based on these measurement methods. Compounds that inhibit this phosphorylation activity are useful as anti-cancer agents having superior specificity for cancer.

TECHNICAL FIELD

The present invention relates to methods for measuring thephosphorylation activity of Cdc7-ASK kinase complex.

BACKGROUND ART

During cell growth, cells repeat cyclic processes whereby they replicatetheir genomic DNA, distribute this DNA equally among daughter cells, andthen divide. This type of cycle is referred to as a cell cycle. The cellcycle can be divided into the G1 phase (DNA synthesis preparatoryphase), S phase (DNA synthesis phase), G2 phase (mitosis preparatoryphase), and M phase (mitotic phase). Cells divide by going through eachstage in order. The progress of the cell cycle is finely regulated bynumerous molecules, controlling unnecessary cell growth. To date, anumber of molecules involved in cell cycle progression have been clonedand functionally analyzed. Abnormalities in molecules involved in cellcycle progression have been reported in numerous cancers, and can beconsidered possible causes of carcinogenesis.

To date, a variety of anti-cancer agents and anticancer agents have beendeveloped. Much abnormal cancer cell growth is recognized as originatingfrom abnormal cell cycle progression. In line with this, anticanceragents that targeting factors which regulate cell cycle progression arebeing vigorously developed. In particular, the function of Cdk-Cyclin,which has been most researched as a target for anticancer agents, hasbeen considerably analyzed. Numerous candidate molecules for anticanceragents that target Cdk-Cyclin have already been reported.

Cdk is a family of molecules with numerous structural similarities, andeach of these molecules forms complexes with various cyclin moleculesspecific to the cell cycle, thus regulating the various stages of thecell cycle. At present, particular molecules that specifically inhibitonly Cdk-Cyclin have not yet been identified from among thesestructurally similar molecules. In other words, obtaining a moleculethat specifically and selectively acts on cancer cells by targetingCdk-Cyclin has been difficult. In addition, although numerous anticanceragents have been found that target regulatory factors in cell divisionor the G1 (stationary) phase, few agents have been discovered thattarget the process of DNA replication, another important regulatorypoint of cell growth.

A serine/threonine kinase different from Cdk-Cyclin has been found toplay an important role at the S phase initiation point (the transitionfrom G1 to S). Namely, in variant line Cdc7, which was isolated as aline with a variant cell division cycle (J. Mol. Biol., 59:183-194,1971), the Cdc7 protein kinase has been demonstrated to function justprior to the initiation of chromosomal DNA replication, and to benecessary for activation of each origin of replication throughout the Sphase (Mol. Cell. Biol., 6:1590-1598, 1986; Genes Dev., 15:480-490,1998; and Genes Dev., 15:491-501, 1998). In addition, yeast Cdc7 kinaseactivity is known to be dependent on regulatory subunit, Dbf4 (Genetics,131:21-29, 1992; and Mol. Cell. Biol., 13:2899-2908, 1993).

Expression of Dbf4 in yeast is cyclical, and regulated at both thetranscription and post-translation levels (Exp. Cell Res., 180:419-428,1989). At least some of the increase in Cdc7 kinase activity during theG1-S transition phase is explained by an increase in expression of Dbf4during the late G1 phase (Mol. Cell. Biol., 13:2899-2908, 1993; and Exp.Cell Res., 180:419-428, 1989). Moreover, since Dbf4 interacts with theorigins of replication in cells (Science, 265:1243-1246, 1994), Cdc7 isthought to trigger S phase initiation by directly activating thereplication apparatus formed on an origin of replication.

The present inventors succeeded in isolating H37 protein, which is ahuman homolog of yeast Dbf4, and DNA encoding it (Unexamined PublishedJapanese Patent Application No. (JP-A) 2000-135090, J. Biol. Chem., Vol.275, No. 37, 29042-29052, 2000). H37 is an important regulatory factorthat governs the switching on and off of DNA replication initiation. H37was subsequently named a human Activator of S phase Kinase (ASK).

ASK is a unique gene in human chromosomes, and its function is essentialfor chromosome replication. Based on the results of developmentalengineering genetic analyses using mice, Cdc7 function was verified asbeing essential at the cellular and animal levels. Cdc7 encodes a kinasecatalyst subunit, while ASK encodes each of the active subunits. The twoform a complex, comprising an active kinase. The activity of Cdc7-ASKkinase is strictly regulated by the cell cycle, and increases in the Sphase when DNA replication occurs. Although this kinase activity ismaintained at a high level throughout the S phase, it is no longerdetected from the M phase to the G1 phase. Regulation of this activitymainly depends on fluctuations in the level of ASK protein expression.Namely, the amount of ASK protein fluctuates during the cell cycle, andas a result, the activity of human Cdc7 kinase, which is regulated bybeing bound to ASK, also fluctuates during the cell cycle.

In addition, the expression level and activity of human Cdc7-ASK istypically observed to increase in actively growing cancer cells. In suchcancer cells, the activity of human Cdc7 kinase activity is thought toincrease with increasing expression of ASK protein. Of even furtherinterest, the present inventors found that the expression level of ASKincreases in response to increases in the growth ability of variouscultured cancer cells.

Subsequent research by the present inventors demonstrated that thespecific substrate phosphorylated by Cdc7-ASK complex is aminichromosome maintenance (MCM) complex (heterohexamer), essential forthe initiation of chromosome replication (J. Biol. Chem., Vol. 275, No.37, 29042-29052, 2000). On the basis of this finding, the presentinventors advocate a model in which Cdc7-ASK phosphorylates the MCMpresent in the replication origin complex, and as a result, inducesreconfiguration of the subunit structure, causing the double-strandedDNA split that is essential for replication initiation.

On the basis of this, phosphorylation of a substrate protein by theCdc7-ASK complex is recognized as being an important phenomenon inregulating the growth of cancer cells. However, many aspects of thisphosphorylation mechanism remain to be elucidated.

DISCLOSURE OF THE INVENTION

An object of the present invention is to clarify the mechanism ofsubstrate protein phosphorylation by the Cdc7-ASK complex, and toprovide methods for evaluating its activity. In addition, based on theseevaluation methods, an object of the present invention is to providemethods for measuring the influence of Cdc7-ASK complex test compoundson the substrate phosphorylation effect. Another object of the presentinvention is to provide substrates, antibodies, or Cdc7-ASK complexesuseful for these methods.

The present inventors considered the Cdc7-ASK complex to be a novel andimportant anti-cancer agent target; that is, they thought thatinhibitors that could inhibit the phosphorylation effect of the Cdc7-ASKcomplex could selectively regulate cancer cell growth, and would becandidates for effective anticancer agents.

In order to evaluate the action of inhibiting the Cdc7-ASK complexphosphorylation effect, it was first essential to establish methods toevaluate this effect. To achieve this, the present inventors firstclarified the mechanism used by the Cdc7-ASK complex to phosphorylatesubstrate proteins. On the basis of this finding, the present inventorsconfirmed that the kinase activity of the Cdc7-ASK complex wasmeasurable, thereby completing the present invention.

The present inventors then discovered that the influence of testcompounds on the Cdc7-ASK complex's substrate protein phosphorylationfunction can be evaluated by applying these methods to measure kinaseactivity. On the basis of this finding, the present inventorsestablished methods for evaluating the inhibitory (or promotional)effect of test compounds on Cdc7-ASK kinase, and methods of screeningfor compounds comprising such effects, thereby completing the presentinvention.

Moreover, while establishing these methods, the present inventorsdiscovered novel substrate compounds, antibodies, Cdc7-ASK complexes orso on useful to these methods, thereby completing the present invention.Namely, the present invention relates to methods for measuring thekinase activity of the Cdc7-ASK complex, as below. Alternatively, thepresent invention relates to methods for evaluating the effect of testcompounds on the kinase activity of the Cdc7-ASK complex, and screeningmethods based on these methods. Moreover, the present invention relatesto substrate compounds, antibodies or Cdc7-ASK complexes useful to thesemethods, or methods for preparing the compounds, antibodies or Cdc7-ASKcomplexes.

[1] A method for measuring the kinase activity of a Cdc7-ASK complex,comprising the following steps:

-   -   (a) contacting a substrate protein with the Cdc7-ASK complex        under conditions that allow phosphorylation of the substrate        protein, wherein the substrate protein is a protein comprising        the amino acid sequence of SEQ ID NO: 1, or a protein        functionally equivalent to that protein;    -   (b) measuring the level of phosphorylation of a serine residue        of the substrate protein at the position corresponding to        position 17 in the amino acid sequence of SEQ ID NO: 1; and    -   (c) measuring the kinase activity of the Cdc7-ASK complex using        the level of phosphorylation as an indicator.

[2] The method according to [1], wherein the level of phosphorylation ismeasured based on the level of binding of an antibody that identifiesthe level of phosphorylation of the serine residue.

8 3] The method according to [1], wherein the Cdc7-ASK complex isderived from a biological sample.

[4] A method for measuring the effects of a test compound on the kinaseactivity of a Cdc7-ASK complex, comprising the following steps:

-   -   (a) contacting a test compound, a substrate protein and a        Cdc7-ASK complex active substance, wherein the substrate protein        is a protein comprising the amino acid sequence of SEQ ID NO: 1,        or a protein functionally equivalent to that protein, and        wherein they are contacted in any of the following orders i) to        iii):        -   i) the test compound and substrate protein are contacted,            followed by contacting the Cdc7-ASK complex active            substance,        -   ii) the substrate protein and Cdc7-ASK complex active            substance are contacted in the presence of the test            compound, or        -   iii) the substrate protein and Cdc7-ASK complex active            substance are contacted, followed by contacting the test            compound;    -   (b) measuring the level of phosphorylation for a serine residue        of the substrate protein at the position corresponding to        position 17 in the amino acid sequence shown in SEQ ID NO: 1;        and    -   (c) measuring the effect of the test compound on the kinase        activity of the Cdc7-ASK complex active substance using the        level of phosphorylation as an indicator.

[5] A method of screening for compounds comprising the effect ofregulating the kinase activity of a Cdc7-ASK complex, comprising thefollowing steps:

-   -   (a) measuring the effect of a test compound on the kinase        activity of the Cdc7-ASK complex according to the method        described in [4]; and    -   (b) selecting a test compound with a high or low level of        phosphorylation by comparison with a control that has not, been        contacted with the test compound.

[6] A screening method according to [5], wherein a compound having a lowlevel of phosphorylation is selected in step (b) of [5].

[7] An inhibitor of cell growth comprising a compound selected accordingto the screening method of [6] as its active ingredient.

[8] A kit for measuring the kinase activity of a Cdc7-ASK complexcomprising:

-   -   (a) a substrate protein comprising a continuous amino acid        sequence that comprises a serine residue at position 17 of the        amino acid sequence of SEQ ID NO: 1, and that which is selected        from the amino acid sequence of SEQ ID NO: 1; and    -   (b) an antibody that identifies the level of phosphorylation of        the serine residue of the substrate protein at the position        corresponding to position 17 of the amino acid sequence of SEQ        ID NO: 1.

[9] A kit for evaluating the effect of a test compound on the kinaseactivity of a Cdc7-ASK complex, comprising:

-   -   (a) a Cdc7-ASK complex active substance; and,    -   (b) a substrate protein comprising a continuous amino acid        sequence that comprises the serine residue at position 17 of the        amino acid sequence of SEQ ID NO: 1, and is selected from this        amino acid sequence.

[10]. A process for producing a Cdc7-ASK complex active substancecomprising the following steps:

-   -   (a) introducing a DNA encoding human Cdc7 protein and a DNA        encoding a protein comprising the amino acid sequence of SEQ ID        NO: 10 or a protein functionally equivalent to the protein, into        prokaryotic cells in a state that allows monocistronic        expression;    -   (b) expressing the two DNAs; and    -   (c) recovering the expressed protein.

[11] An antibody that identifies the level of phosphorylation of theserine residue at position 17 of a protein comprising the amino acidsequence of SEQ ID NO: 1.

[12] A protein according to any of the following (a) to (d):

-   -   (a) a protein comprising the amino acid sequence of SEQ ID NO:        1;    -   (b) a protein comprising a continuous amino acid sequence that        is selected from the amino acid sequence of SEQ ID NO: 3, and        comprises the serine of position 17;    -   (c) a protein comprising an amino acid sequence in which one or        more amino acids in the amino acid sequence of SEQ ID NO: 1 are        substituted, deleted, added and/or inserted, wherein the protein        is phosphorylated by human Cdc7-ASK complex; and    -   (d) a protein comprising an amino acid sequence comprising 90%        or more homology with the amino acid sequence of SEQ ID NO: 3,        wherein the protein is phosphorylated by human Cdc7-ASK complex.

[13] A protein comprising a continuous amino acid that comprises theamino acid sequence of SEQ ID NO: 10 and that which is selected from theamino acid sequence of SEQ ID NO: 9.

[14] A polypeptide according to [13] that comprises the amino acidsequence of SEQ ID NO: 10.

Alternatively, the present invention relates to methods for inhibitingcell growth that comprise the step of administering a compound selectedaccording to the screening method of [6]. In addition, the presentinvention relates to the use of a compound, selected according to thescreening method of [6], in the production of a cell growth inhibitor.

The present invention relates to methods for measuring the kinaseactivity of Cdc7-ASK complex, where the methods comprise the followingsteps:

-   -   (a) contacting a substrate protein with the Cdc7-ASK complex        under conditions that allow phosphorylation of the substrate        protein, wherein the substrate protein is a protein comprising        the amino acid sequence of SEQ ID NO: 1, or a protein        functionally equivalent to that protein;    -   (b) measuring the level of phosphorylation of a serine residue        of the substrate protein at the position corresponding to        position 17 in the amino acid sequence of SEQ ID NO: 1; and    -   (c) measuring the kinase activity of the Cdc7-ASK complex using        the level of phosphorylation as an indicator.

In the present invention, a protein comprising the amino acid sequenceof SEQ ID NO: 1, or a protein functionally equivalent to that protein,is used as a substrate protein.

The structure of MCM2 protein is known. The amino acid sequence of humanMCM2, and the nucleotide sequence of the CDNA that encodes it, are shownin SEQ ID NOS: 2 and 3 respectively. Cdc7-ASK complex phosphorylates aspecific amino acid residue of the human MCM2 of SEQ ID NO: 3. Thepresent inventors have previously demonstrated that Cdc7-ASK complexphosphorylates MCM complex or free MCM2. However, that the Cdc7-ASKcomplex specifically phosphorylates the serine at position 17 from theMCM2 N-terminal is a novel finding, revealed by the present inventors.

Thus, proteins comprising continuous amino acid sequences that comprisethe position 17 serine and are selected from the amino acid sequence ofSEQ ID NO: 3, can be used as substrate proteins in the presentinvention, as long as they can be phosphorylated by Cdc7-ASK complex.Examples of other substrate proteins that can be used in the presentinvention are indicated below. Tags can be added to these substrateproteins. In addition, these substrate proteins can be bound to a solidphase, as described later.

-   -   (a) a protein comprising the amino acid sequence of SEQ ID NO:        1;    -   (b) a protein comprising a continuous amino acid sequence that        is selected from the amino acid sequence of SEQ ID NO: 3, and        comprises the serine of position 17;    -   (c) a protein comprising an amino acid sequence in which one or        more amino acids in the amino acid sequence of SEQ ID NO: 1 are        substituted, deleted, added and/or inserted, wherein the protein        is phosphorylated by human Cdc7-ASK complex; and    -   (d) a protein comprising an amino acid sequence comprising 90%        or more homology with the amino acid sequence of SEQ ID NO: 3,        wherein the protein is phosphorylated by human Cdc7-ASK complex.

In the present invention, the phosphorylation of a certain protein bythe Cdc7-ASK complex can be confirmed based on the results of, forexample, the reactions described in Examples. Namely, a phosphatecompound and the Cdc7-ASK complex are incubated with a substrate proteinunder conditions that allow phosphorylation of that protein (describedbelow). Next, the level of phosphorylation at the serine residue at theposition corresponding to position 17 in the amino acid sequence shownin SEQ ID NO: 1 is measured. When there is no significant differenceobserved between the measured level of phosphorylation and the levelwhen, for example, a protein comprising the amino acid sequence of SEQID NO: 1 is used as a substrate protein, then that protein is judged tobe a protein phosphorylated by human Cdc7-ASK complex.

The number of amino acids that make up the substrate protein of thepresent invention can generally be five or more, usually ten or more,and preferably 50 or more. In addition, although there are norestrictions as to the size of the substrate protein, it can be a shortfragment of, for example, 400, generally 300, or 200 or less.Phosphorylation by the Cdc7-ASK complex can be observed morespecifically using a short fragment as a substrate protein.

In the present invention, proteins comprising, for example, the aminoacid sequence of SEQ ID NO: 1, can be indicated as preferable substrateproteins. The amino acid sequence of SEQ ID NO: 1 is a fragment sequencecomprising 130 amino acids on the N-terminal side of the human MCM2 ofSEQ ID NO: 3. The serine located at the 17th amino acid counting fromN-terminal is phosphorylated by the Cdc7-ASK complex.

In addition, proteins which are phosphorylated by human Cdc7-ASK complexand that comprise amino acid sequences in which one or more amino acidsin the amino acid sequence of SEQ ID NO: 1 are substituted, deleted,added and/or inserted, can also be used as substrate proteins. In thepresent invention the number of amino acids subjected to mutation is,for example, one to ten, preferably one to six, and more preferably oneto three.

Mutation of an amino acid sequence may be a mutation that is inducedartificially, or that occurs spontaneously. When substituting aminoacids, conservative substitution can be used.

In general, to maintain protein function, the substitute amino acids arepreferably those with properties similar to the amino acids prior tosubstitution. This type of amino acid substitution is referred to asconservative substitution.

For example, since Ala, Val, Leu, Ile, Pro, Met, Phe and Trp are allclassified as non-polar amino acids, they have mutually similarproperties. Examples of non-charged amino acids include Gly, Ser, Thr,Cys, Tyr, Asn and Gln. Alternatively, examples of acidic amino acidsinclude Asp and Glu. Moreover, examples of basic amino acids includeLys, Arg and His. The amino acids that compose each of these groups havemutually similar properties. Consequently, there is a high possibilitythat protein function will be maintained when these amino acids aresubstituted with other amino acids from the same group.

This type of protein can be obtained by introducing a mutation into thenucleotide sequence of the cDNA of the human MCM2 of SEQ ID NO: 1.Technology for introducing mutations into genes comprising knownnucleotide sequences is known. Alternatively, proteins comprisingdesired amino acid sequences can be prepared by chemical synthesis.

In the substrate proteins of the present invention, the serine locatedat the position corresponding to position 17 of the amino acid sequenceof SEQ ID NO: 1 is important as the phosphorylation target of theCdc7-ASK complex. Thus, even when the substrate protein is a proteincomprising an amino acid sequence mutation, it is important that aserine be preserved at position 17, or a position homologous to position17. The term “position homologous to position 17” refers to a positionthat corresponds to position 17 of the amino acid sequence of SEQ ID NO:1, when a given amino acid sequence is aligned with the amino acidsequence of SEQ ID NO: 1. Multiple methods are known for aligning aminoacid sequences. For example, a wide variety of software, based onalgorithms such as the BLAST algorithm, is in practical use for aligningdifferent amino acid sequences.

In addition, a protein that comprises an amino acid sequence comprising90% or more homology to the amino acid sequence of SEQ ID NO: 3, andwhich is phosphorylated by human Cdc7-ASK complex, can also be used as asubstrate protein of the present invention. For example, human Cdc7-ASKcomplex phosphorylates not only human MCM2, but also mouse MCM2. Thus,mouse MCM2 can be used as a substrate protein in the present invention.The amino acid sequence of mouse MCM2 is shown in SEQ ID NO: 5, whilethe nucleotide sequence that encodes this amino acid sequence is shownin SEQ ID NO: 4. Mouse MCM2 and human MCMC2 are 99% homologous. Thus,proteins-comprising 99% or more homology to the amino acid sequence ofhuman SEQ ID NO: 3 (the full-length amino acid sequence of human MCM2)are useful as substrate proteins of the present invention.

In the present invention, the level of phosphorylation of the serine atposition 17 of human MCM2, or of a serine at a position homologous tothis serine, is measured as an indicator. When using a protein thatcomprises an amino acid sequence different from the amino acid sequencesof SEQ ID NOs: 1 and 3 as a substrate protein, it is important topreserve the serine at position 17 of SEQ ID NO: 1, or serines atpositions homologous to position 17. For example, in mouse MCM2, theserine at position 26 is the serine at a position homologous to position17. Thus, when using mouse MCM2 fragments as substrate proteins, it ispreferable to use proteins that comprise an amino acid sequencecomprising a serine at position 26.

The substrate protein of the present invention can be obtained based ongenetic engineering techniques. Namely, a protein having the desiredamino acid sequence can be obtained by expressing a DNA encoding MCM2 ina suitable expression system. The nucleotide sequence of a DNA encodingMCM2 is shown in SEQ ID NO: 2.

For example, the Examples show an example of expressing the MCM2 geneusing Escherichia coli. The expression product can be purified usingtechniques such as salting out, gel filtration, ion exchangechromatography or so on. Purification by affinity chromatography, usingthe binding affinity of a tag with binding affinity that has been fusedto MCM2, can also be used. Examples of binding tags that can be usedinclude a histidine tag comprising several histidines (His-Tag),β-D-galactosidase, glutathione S-transferase (GST), thioredoxin,maltose-binding protein, Myc, Xpress, and FLAG. If GST is used, forexample, the expressed protein can be purified easily with a GlutathioneSepharose 4B column or so on.

Depending on the expression system, MCM2 may be recovered in aphosphorylated state. When the substrate protein has been phosphorylatedin the expression system, the protein can be dephosphorylated bytreatment with an enzyme such as phosphatase, which acts on phosphategroups. The measurement methods of the present invention comprise thestep of measurement using the phosphorylation of substrate proteins asan indicator. Thus, it is particularly preferable that the substrateprotein sites to be phosphorylated by the Cdc7-ASK complex are notphosphorylated. In addition, methods are also known for chemicallysynthesizing proteins comprising the desired amino acid sequence.

The present invention's methods for measuring the kinase activity ofCdc7-ASK complex comprise the step of contacting the aforementionedsubstrate proteins with samples whose Cdc7-ASK complex kinase activityis to be measured, under conditions that allow phosphorylation of thesubstrate protein. In the present invention, “conditions that allowphosphorylation of the substrate protein” refer to conditions suited toexpression of Cdc7-ASK complex kinase activity. More specifically, thetemperature, salt concentration, and pH are suited to the expression ofenzyme activity, and phosphate compounds are present for substrateprotein phosphorylation. “Conditions suitable for enzyme activity” canbe exemplified as below. Namely, such reaction conditions can beimparted by contacting the aforementioned components in HEPES bufferadjusted to pH 7.0 to 7.5.

In the present invention, “conditions that allow phosphorylation of asubstrate protein” refer to contacting a sample with a substrate proteinin the presence of a compound comprising a phosphate group necessary forsubstrate protein phosphorylation. Phosphate groups can be supplied by,for example, coexisting with adenosine triphosphate (hereinafterdescribed as ATP).

The substrate protein and phosphate compound that comprise theaforementioned reaction are preferably added in excess with regards tothe Cdc7-ASK complex kinase activity in the sample. It is generallydifficult to accurately predict the kinase activity of Cdc7-ASK complexcontained in a sample. However, those with ordinary skill in the art areable to experimentally predetermine an adequate amount of substratecompound or phosphate compound, such that the components required by thereaction are sufficient for enzyme reaction. For example, when measuringa human-derived biological sample, the amount of substrate protein to beused is, for example, 0.1 μg or more, and generally 0.2 μg to 1 μg. Theamount of phosphate compound to be added as ATP can be, for example,0.01 mM or more, and generally 0.1 mM to 1 mM. Furthermore, phosphatecompound is added in an amount that is necessary and sufficient forphosphbrylation of the substrate protein used in the reaction system.

The reaction time for phosphorylation is typically ten minutes or more,and preferably 30 minutes or more. Reliable phosphorylation of substrateprotein can be expected from, for example, 50 to 100 minutes ofreaction. The time required for phosphorylation varies according to theamount of substrate protein used, and the level of Cdc7-ASK complexactivity in the reaction solution. Those with ordinary skill in the artare able to suitably set the reaction time required for phosphorylationunder given conditions.

In the measurement methods of the present invention, the level ofsubstrate protein phosphorylation over a predetermined time relates tophosphorylation activity in the sample. Alternatively, thephosphorylation activity in a sample can also be measured by using, asan indicator, the reaction time required to phosphorylate a substrateprotein to a predetermined level. Namely, the greater a sample'sphosphorylation activity, the faster the substrate proteinphosphorylation will reach a predetermined level. Moreover,phosphorylation activity in the sample can also be measured by using theamount of sample required to phosphorylate the substrate protein to apredetermined level as an indicator. In this embodiment, the amount ofsample required for phosphorylation is reversely proportional to thelevel of phosphorylation activity in the sample.

When the phosphorylation activity in a sample far exceeds thatpredicted, the majority of the substrate protein will be phosphorylatedin a short period of time. If this happens, accurate evaluation ofphosphorylation activity may be impossible. Thus, when such results areobtained, it is preferable to reduce the amount of sample added to thereaction, and remeasure phosphorylation activity.

The measurement methods of the present invention comprise the step ofmeasuring the phosphorylation level of the substrate protein for theserine residue at the position corresponding to position 17 of the aminoacid sequence of SEQ ID NO: 1. The level of phosphorylation at thisposition can be measured immunologically by using, for example, anantibody that recognizes the phosphorylated state of the serine residueat a position corresponding to position 17.

More specifically, an antibody can be used for which binding activitydecreases with serine dephosphorylation, as compared to binding activityto an antigen-determining group comprising the phosphorylated serineresidue of a protein with an amino acid sequence comprising a serinethat corresponds to position 17. Conversely, antibodies that bind toantigen-determining groups comprising a non-phosphorylated serineresidue, but for which bonding activity is decreased withphosphorylation of the serine residue, can also be used.

In the measurement methods of the present invention, antibodies thatidentify the level of phosphorylation of the aforementioned serineresidue, used to measure the level of phosphorylation, can be obtainedin, for example, the following manner: First, a synthetic peptide thatcomprises a continuous amino acid sequence comprising the applicableserine residue can be used as the immunogen. The length of the syntheticpeptide used as the immunogen can be at least three or more, for examplefive or more, normally ten or more, and preferably ten to 20 aminoacids. A preferable example of an immunogen is an amino acid sequencecomposed of 15 continuous amino acids, containing serine at position 17,and selected from the amino acid sequence described in SEQ ID NO: 1. Theposition of the serine can be arbitrary. For example, in the Examples, asynthetic peptide was used as an immunogen that comprised an amino acidsequence in which serine was located in the center of 15 amino acids.The synthetic peptide can be bonded to a carrier protein. Keyhole Limpethemocyanin and such can be used as the carrier protein.

The serine residue corresponding to position 17 in the immunogen can bephosphorylated in advance. Methods for obtaining synthetic peptides inwhich a specific amino acid has been phosphorylated are known. When theserine in the immunogen is phosphorylated, antibodies comprising theactivity of binding to phosphorylated serine can be obtained.

Immunogens are administered to immunity animals by mixing with suitableadjuvants. Immunization is normally performed several times, and bloodis collected from immunized animals after confirming an adequate rise inantibody titer. Serum recovered from the collected blood can be used asantiserum that contains an antibody of the present invention. Purifiedantibodies can be obtained by purifying immunoglobulins from suchantiserums.

Alternatively, cell lines that produce monoclonal antibodies can beestablished by immortalizing antibody-producing cells from the immunizedanimals, and then selecting clones that produce antibodies comprisingthe desired reactivity. For example, antibody-producing cells can beimmortalized by fusion with cells such as myeloma cells, to producehybridomas.

Antibodies can be confirmed to have desired reactivity by using thesynthetic peptide used as the immunogen. For example, antibodies thatrecognize phosphorylated substrate proteins can be obtained by selectingantibodies that bind to synthetic peptides comprising phosphorylatedserine. Moreover, an antibody that specifically binds to aphosphorylated substrate protein can be obtained by removing from thisantibody any antibodies that bind to synthetic peptides that comprisenon-phosphorylated serine. Using this kind of selection process,antibodies that recognize the phosphorylated state of substrateproteins, essential to the present invention, can be obtained bytreating with antiserum or purified antibody.

Hybridomas that produce monoclonal antibodies able to recognize thephosphorylated state of substrate proteins can be selected by screeningantibodies produced by the hybridomas, according to similar selectionprocesses.

In the measurement methods of the present invention, the aforementionedantibodies can be labeled in advance. Antibodies bound to phosphorylatedsites can be easily detected by labeling antibodies. In addition, if thesignals from labels can be amplified, measurements of higher sensitivitycan be expected.

Arbitrary labeling components can be used for antibody labeling. Forexample, fluorescent dyes, enzymes, radioactive substances, and so oncan be used as labels. In addition, substances with high electrondensity, such as ferritin and gold colloids, can also be used as labels.

Examples of fluorescent dyes include fluorescein isothiocyanate (FITC),rhodamine β-isothiocyanate (RITC), and phycoerythrin (PE). Examples ofenzymes include peroxidase, β-D-galactosidase, microperoxidase, alkalinephosphatase, acidic phosphatase., and cytochrome c. Examples ofradioactive substances include ¹²⁵I, ¹⁴C and ³H. Methods for labelingantibodies with these labeling components are known.

In addition, antibodies can also be labeled using antibodies(hereinafter referred to as secondary antibodies) that bind tophosphorylation-recognizing antibodies (primary antibodies). Forexample, a phosphorylation-recognizing. antibody is contacted with asubstrate protein, and then reacted with a secondary antibody. Theamount of secondary antibody indirectly bound to the substrate ismeasured, and the amount of bound primary antibody is then determinedfrom the measured amount of secondary antibody. Thus, the level ofsubstrate phosphorylation can be determined. For example, when using amouse monoclonal antibody as a primary antibody, anti-mouse IgG goatantibody can be used as a secondary antibody.

Alternatively, a primary antibody can also be indirectly labeled bylabeling with a substance with binding affinity, and then using theaffinity of a binding partner to that affinitive substance. For example,if a primary antibody is avidinyllated, a biotinylated enzyme can bebound to the antibody using the affinity between avidin and biotin.

In the measurement methods of the present invention, Cdc7-ASK substrateprotein can be used in an immobilized state by pre-binding it to aninsoluble support. Immobilizing the substrate protein makes it easier todetect binding with the aforementioned phosphorylation-recognizingantibodies. In other words, bound and unbound antibodies are easilyseparated by contacting the antibodies with a substrate protein,removing the liquid phase, and washing the solid phase. The amount ofantibody bound (or not bound) to the substrate protein can then beeasily measured by measuring the antibody in the solid phase (or liquidphase). Immobilizing substrate proteins on a solid phase alsocontributes to their stabilization.

Insoluble supports used for immobilizing substrate proteins includeresins such as polystyrene resins, polycarbonate resins, silicon resinsand Nylon resins, glass, and micelle particles, and the material used isnot particularly restricted. There are also no particular restrictionson the form of the insoluble support, and examples of forms that can beused include trays, spheres, rods, fibers, cells and test tubes. Thesubstrate proteins can be immobilized on insoluble supports by physicaladsorption or chemical bonding. The insoluble support on which asubstrate protein has been immobilized can also be blocked as necessary.Albumin, skim milk, and so on are used for blocking. Blocking theinsoluble support inhibits non-specific antibody binding. In addition,blocking can also be expected to exhibit a protective effect on thesubstrate protein.

In the present invention, the amount of antibody bound (or not bound) toa substrate protein is correlated with a sample's Cdc7-ASK complexkinase activity. More specifically, when using, for example, an antibodythat binds to an antigen-determining group comprising a substrateprotein's phosphorylated serine, the amount of antibody bound to thesubstrate protein is proportional to the degree of kinase activity ofthe Cdc7-ASK complex in the sample. The kinase activity in a sample canalso be quantitatively determined by comparison with the results ofmeasurements obtained from a Cdc7-ASK complex standard, for which thelevel of kinase activity is already known.

The present invention measures the kinase activity of Cdc7-ASK based onthe phosphorylation state of a substrate protein region phosphorylatedby Cdc7-ASK. Thus, the kinase activity of Cdc7-ASK can be specificallymeasured, without being affected by other substances that comprisephosphorylation activity and which may coexist in the sample. As aresult, the state of cell growth can be specifically detected bymeasuring Cdc7-ASK kinase activity based on the methods of the presentinvention.

Cdc7-ASK maintains a high level of activity, particularly in the S phaseof the cell cycle, and its activity decreases in the G1 phase. Thus,measuring the activity of Cdc7-ASK kinase is useful as an indicator of acell's transition from the G1 phase to the S phase. The S phase is theperiod when cells proceed to replicate nucleic acids in preparation forcell growth. Thus, for example, finding a large number of cells in the Sstage among the cells in a certain biological sample means that cellgrowth is progressing in that sample. More specifically, if a largenumber of cells with high-level Cdc7-ASK kinase activity are found in acancer tissue by using a measurement method of the present invention,this cancer tissue contains many actively growing cells. In other words,it is possible that the cancer is highly malignant.

In addition, the present invention relates to methods for measuring theeffect of a test compound on the kinase activity of Cdc7-ASK complex,comprising the following steps:

-   -   (a) contacting a test compound, a substrate protein and a        Cdc7-ASK complex active substance, wherein the substrate protein        is a protein comprising the amino acid sequence of SEQ ID NO: 1,        or a protein functionally equivalent to that protein, and        wherein they are contacted in any of the following orders i) to        iii):        -   i) the test compound and substrate protein are contacted,            followed by contacting the Cdc7-ASK complex active            substance,        -   ii) the substrate protein and Cdc7-ASK complex active            substance are contacted in the presence of the test            compound, or        -   iii) the substrate protein and Cdc7-ASK complex active            substance are contacted, followed by contacting the test            compound;    -   (b) measuring the level of phosphorylation for a serine residue        of the substrate protein at the position corresponding to        position 17 in the amino acid sequence shown in SEQ ID NO: 1;        and    -   (c) measuring the effect of the test compound on the kinase        activity of the Cdc7-ASK complex active substance using the        level of phosphorylation as an indicator.

In the aforementioned method, the term “Cdc7-ASK complex activesubstance” refers to a complex that is functionally equivalent to theCdc7-ASK complex. More specifically, a “Cdc7-ASK complex activesubstance” is defined as a complex comprising the function ofphosphorylating a substrate protein serine that is to be phosphorylated.Thus, if it makes up a complex that comprises this activity, the complexcan still be used as a Cdc7-ASK complex active substance of the presentinvention, even if the Cdc7 or ASK comprising the complex comprisestructures different to that of human-derived proteins.

Whether a certain protein complex comprises the function ofphosphorylating a substrate protein serine to be phosphorylated can beconfirmed based on, for example, a method such as that shown in theExamples. Namely, the aforementioned substrate protein and proteincomplex are incubated under conditions that allow phosphorylation of thesubstrate protein. If there is no significant difference observed whencomparing the substrate protein phosphorylation level after thisincubation with the level after incubation with human Cdc7-ASK complex,then that protein complex is judged to be functionally equivalent toCdc7-ASK complex.

The structures of human-derived Cdc7 and ASK are known. The nucleotidesequence of a cDNA encoding human Cdc7 is shown in SEQ ID NO: 6, whilean amino acid sequence of human Cdc7 is shown in SEQ ID NO: 7. Inaddition, the nucleotide sequence of a cDNA encoding human ASK is shownin SEQ ID NO: 8, while an amino acid sequence of human ASK is shown inSEQ ID NO: 9. With regards to these known Cdc7 and ASK, for example,proteins such as the following can be used for each subunit composing aCdc7-ASK complex active substance of the present invention: In thepresent invention, those subunits that differ from naturally-occuringhuman Cdc7 or human ASK, but are capable of making up Cdc7-ASK complexactive substances comprising kinase activity, are respectively referredto as Cdc7 subunits and ASK subunits.

First, proteins that can be used as the Cdc7 subunit are those thatcompose a complex comprising the effect of phosphorylating a substrateprotein by forming a complex with an aforementioned human ASK, whereinthey are encoded by the following polynucleotides:

-   -   (a) a polynucleotide that comprises a region that encodes the        nucleotide sequence of SEQ ID NO: 6;    -   (b) a polynucleotide that comprises a nucteotide sequence that        encodes the amino acid sequence of SEQ ID NO: 7;    -   (c) a polynucleotide that encodes a protein that comprises an        amino acid sequence in which one or more amino acids are        substituted, deleted, added and/or inserted in the amino acid        sequence of SEQ ID NO: 7, in which the polynucleotide composes a        complex having phosphorylation effect on a substrate protein by        forming a complex with the aforementioned human ASK; and,    -   (d) a polynucleotide that hybridizes with a polynucleotide that        contains a region that encodes the nucleotide sequence of SEQ ID        NO: 6 under stringent conditions, in which the polynucleotide        encodes a protein that composes a complex having phosphorylation        effect on a substrate protein by forming a complex with the        aforementioned human ASK.

In the present invention, a protein encoded by an aforementionedpolynucleotide can be confirmed to make up a complex comprising theeffect of phosphorylating a substrate protein by forming a complex withan aforementioned human ASK in the following manner: Namely, theaforementioned polynucleotide is first co-expressed in suitable hostcells along with a DNA comprising a nucleotide sequence encoding humanASK. The phosphorylation activity of the resulting expression product isthen evaluated by, for example, a method such as that described in theExamples, and then compared with Cdc7-ASK phosphorylation activity. As aresult, if there is no significant difference observed between the twophosphorylation activities, the Cdc7 subunit making up the complex isjudged to be a protein that comprises a complex comprising the effect ofphosphorylating a substrate protein by forming a complex with theaforementioned human ASK.

On the other hand, proteins encoded by the following polynucleotides andcomprising complexes that comprise the effect of phosphorylatingsubstrate proteins by forming complexes with the aforementioned humanCdc7, can be used as ASK subunits that comprise Cdc7-ASK complex activesubstances of the present invention:

-   -   (a) a polynucleotide that comprises a region that encodes the        nucleotide sequence of SEQ ID NO: 8;    -   (b) a polynucleotide that comprises a nucleotide sequence that        encodes the amino acid sequence of SEQ ID NO: 9;    -   (c) a polynucleotide that encodes a protein that comprises an        amino acid sequence in which one or more amino acids are        substituted, deleted, added and/or inserted in the amino acid        sequence of SEQ ID NO: 9, in which the polynucleotide composes a        complex having phosphorylation effect on a substrate protein by        forming a complex with the aforementioned human Cdc7; and,    -   (d) a polynucleotide that hybridizes with a polynucleotide that        comprises a region that encodes the nucleotide sequence of SEQ        ID NO: 8 under stringent conditions, in which the polynucleotide        encodes a protein that composes a complex having phosphorylation        effect on a substrate protein by forming a complex with the        aforementioned human Cdc7.

In the present invention, proteins encoded by the aforementionedpolynucleotides can be confirmed to make up complexes comprising theeffect of phosphorylating substrate proteins by forming complexes withthe aforementioned human Cdc7 in the following manner: Namely, anaforementioned polynucleotide is first co-expressed in suitable hostcells along with a DNA comprising a nucleotide sequence that encodeshuman Cdc7. The phosphorylation activity of the resulting expressionproduct is then evaluated by, for example, a method such as thatdescribed in the Examples, and then compared with Cdc7-ASKphosphorylation activity. As a result, if there is no significantdifference observed between the phosphorylation activities of the two,the ASK subunit making up the complex is judged to be a protein thatcomprises a complex comprising the effect of phosphorylating a substrateprotein by forming a complex with the aforementioned human Cdc7.

For example, the present inventors clearly demonstrated that a proteincomprising an amino acid sequence corresponding to positions 174 to 349from the N-terminal in the 674 amino acid sequence that comprises humanASK is a region essential to the formation of a complex with Cdc7 andfor expression of kinase activity. An amino acid sequence that comprisesthis region is shown in SEQ ID NO: 10. Such human ASK protein fragmentscan be used as ASK subunits that compose the Cdc7-ASK complex activesubstance of the present invention, as long as they comprise theaforementioned activity.

In the present invention, polynucleotides that encode the aforementionedCdc7 subunits or ASK subunits can be obtained by PCR or hybridizedscreening from an arbitrary cDNA library. Human libraries as well asthose from mammals other than humans such as mice or rats, or eukaryoticcells such as Caenorhabditis and Schizosaccharomyces, can be used as thecDNA libraries.

The Cdc7-ASK complex active substances of the present invention can beextracted from cells or can be produced using genetic engineeringtechniques. Production methods using genetic engineering techniques arepreferable since they allow a large amount of protein to be easilyobtained. A complex comprising human Cdc7 and ASK expressed in insectcells, for example, can be used as a Cdc7-ASK complex active substanceof the present invention.

For example, the present inventors have already succeeded in producingsuch complexes (J. Biol. Chem., Vol. 275, No. 37, 29042-29052, 2000).More specifically, the Cdc7-ASK complex active substances used in thepresent invention can be obtained by using insect cells in accordancewith, for example, the following steps (a) to (c):

-   -   (a) introducing a DNA encoding a human Cdc7 subunit and a DNA        encoding human ASK subunit into insect cells;    -   (b) co-expressing the aforementioned two types of introduced DNA        in the aforementioned insect cells; and,    -   (c) purifying the expressed protein (complex).

Any polynucleotide described in the aforementioned (a) to (e) can beused for a DNA that encodes a human Cdc7 subunit used in theaforementioned step (a). Their forms are not particularly restricted,and include cDNA, genomic DNA and synthetic DNA. For example, DNAscomprising the nucleotide sequence shown in SEQ ID NO: 6 can be used asDNAs that encode a human Cdc7 subunit.

Similarly, any polynucleotide described in the aforementioned (a) to (e)can be used as a DNA that encodes a human ASK subunit used in theaforementioned step (a). For example, a DNA comprising the nucleotidesequence shown in SEQ ID NO: 8 can be used as a DNA that encodes a humanASK protein.

Sf9 cells or Sf21 cells derived from Spodoptera frugiperda, or Tn5 cellsderived from Trichoplusiani, can be used as the insect cells. Thesecells are also commercially available (Invitrogen or Pharmingen), andcan also be obtained from ATCC. DNAs encoding a Cdc7 subunit and a ASKsubunit can be introduced into insect cells using a baculovirus. Forexample, a recombinant virus is produced by homologous recombination bysubcloning these DNAs into a transfer vector and simultaneouslytransfecting insect cells with the resulting plasmid and baculovirusDNA. This recombinant virus infects insect cells and co-expresses theCdc7 subunit and ASK subunit within the insect cells. In order toincrease the amount of protein expressed, the recombinant virus ispreferably purified and amplified prior to expression of the protein.

Examples of transfer vectors that can be used include commerciallyavailable pVL1392 (Pharmingen), pPAK8 (Clontech), pAcUW51 (Pharmingen),pAcUW31 (Clontech), and pAcAB3 (Pharmingen). Transfer vectors capable ofsimultaneously subcloning a Cdc7 subunit and an ASK subunit areparticularly preferable as transfer vectors. For example, two genes canbe subcloned by using a transfer vector having two promoters, such aspAcUW51 (Pharmingen), or a transfer vector having three promoters, suchas pAcAB3 (Pharmingen). Alternatively, different transfer vectors thatrespectively subclone the Cdc7 subunit and ASK subunit can also be used.

In addition, examples of baculovirus DNA that can be used includeBaculoGold™ Linearized Baculovirus DNA (Pharmingen) and wild typeBaculovirus AcNV DNA (Pharmingen, Invitrogen). Various types of kit arecommercially available for expression systems using baculovirus, and anyof these kits may be used.

The Cdc7 subunit and ASK subunit genes, which are exogenous genes, canbe co-expressed in insect cells using the aforementioned transfervectors and baculoviruses. The Cdc7-ASK complex active substances usedin the present invention can be obtained by culturing transformed cellsunder conditions that allow expression, and then recovering theexpression product. To facilitate recovery, suitable tags can be fusedto either Cdc7 or ASK or both.

A Cdc7- and ASK-subunit complex can be also obtained in prokaryoticcells, according to the methods described below. For example, when usingEscherichia coli as the prokaryotic cells, a Cdc7-ASK kinase complexactive substance can be prepared according to the following steps:

-   -   (A) introducing a DNA encoding human Cdc7 subunit and a DNA        encoding a protein comprising the amino acid sequence described        in SEQ ID NO: 10 or a protein functionally equivalent to the        protein, into Escherichia coli in a state that allows        monocistronic expression;    -   (B) expressing the two DNAs; and    -   (C) recovering the expressed protein.

According to the present inventors' studies, in an Escherichia colisystem, inserting and expressing a partial ASK (SEQ ID NO: 10), which isnot the full length human ASK protein, eventually enabled acquisition ofa Cdc7-ASK complex active substance that comprised kinase activity. Inthe present invention, a protein functionally equivalent to a proteincomprising the amino acid sequence of SEQ ID NO: 10 refers to a proteinthat can form a complex with a Cdc7 subunit and express kinase activity.

Whether or not a certain protein expresses kinase activity by composinga complex with a Cdc7 subunit can be confirmed as follows: Namely, a DNAthat encodes the protein is first co-expressed in suitable host cellsalong with a DNA comprising a nucleotide sequence that encodes humanCdc7. The phosphorylation activity of the resulting expression productis then evaluated by, for example, a method such as that described inthe Examples, and this is then compared with the phosphorylationactivity of Cdc7-ASK. As a result, if no significant difference isobserved between the two phosphorylation activities, the ASK subunitthat makes up the complex is judged to be a protein that makes up acomplex comprising the effect of phosphorylating a substrate protein byforming a complex with the aforementioned human Cdc7.

Proteins that retain the aforementioned activity and comprise homologyof 90% or more with the amino acid sequence of SEQ ID NO: 10 can beindicated as functionally equivalent proteins. In addition, proteinscomprising an amino acid sequence in which one or more amino acids aresubstituted, deleted, added and/or inserted in the amino acid sequenceof SEQ ID NO: 10 that retain the aforementioned activity are included inthe functionally equivalent proteins. The number of mutated amino acidsis normally 20 or less, for example ten or less, and preferably five orless, for example, one to three. Amino acids can be substituted byconservative substitution. Proteins in which a tag has been fused to theamino acid sequence of SEQ ID NO: 10 are included in the functionallyequivalent proteins of the present invention.

Step (A) uses a DNA encoding a human Cdc7 subunit similar to thatpreviously described, and a DNA encoding a fragment sequence of a humanASK subunit (hereinafter abbreviated as “partial ASK”). A proteincomprising the amino acid sequence shown in SEQ ID NO: 10, or a proteinfunctionally equivalent to the protein, is used as the proteincomprising the fragment sequence. The amino sequence of SEQ ID NO: 10 isa partial sequence corresponding to the amino acid sequence from the174th to the 349th amino acids of SEQ ID NO: 9. The amino acid sequencedescribed in SEQ ID NO: 11 is encoded by the nucleotide sequencedescribed in SEQ ID. NO: 10. The nucleotide sequence described in SEQ IDNO: 10 is a partial nucleotide sequence corresponding to the 1027th tothe 1564th nucleotide of the nucleotide sequence of SEQ ID NO: 8.Protein fragments comprising this amino acid sequence are regionsnecessary for the expression of kinase activity by forming a complexwith human Cdc7. Herein, as long as a partial ASK encodes a partial ASKprotein, it is not limited to this sequence, and DNAs comprising anarbitrary nucleotide sequence that takes codon degeneracy into accountare included. The form of this DNA is also unrestricted, and includescDNA, genomic DNA and synthetic DNA.

In the present invention, the term “a state that allows monocistronicexpression” refers to transcribing a DNA encoding a Cdc7 subunit and aDNA encoding an ASK subunit as a single mRNA molecule, and translatingthem in to two protein molecules. In order to allow monocistronicexpression, these DNAs are arranged so as to enable expression under theregulation of a common transcription regulatory region. They aredesigned so that a nucleotide sequence such as a terminator, whichterminates transcription, is not contained between the two genes. Thetwo genes are preferably arranged in close proximity. However, if thetwo translation frames are consecutive, the genes will be translated asa single fusion protein, and thus a stop codon is arranged between thetwo genes.

In addition, arranging a ribosome binding sequence (RBS; Shine-Dalganosequence) between the two genes is effective in facilitating efficienttranslation of each of the two mRNA protein-encoding regions. In theExamples, a construct is indicated for monocistronic expression inEscherichia coli of human Cdc7 and human partial ASK protein comprisingthe amino acid sequence of SEQ ID NO: 10. The vectors used to producethe construct are not limited. More specifically, by sequentiallycloning, for example, DNAs encoding a Cdc7 subunit and an ASK subunitinto the cloning site of a commercially available vector such aspGEX-2T, as indicated in the Examples, a vector that allows theirmonocistronic expression can be obtained.

A desired Cdc7-ASK complex active substance can be obtained bytransforming this vector construct in Escherichia coli and recoveringthe expression product from the culture, according to establishedmethods.

The expression product can be purified by a technique such as saltingout, gel filtration, or ion exchange chromatography. In methods forproducing a complex that use these genetic engineering techniques, a tagcan be fused to either or both of the subunits that comprise thecomplex. Purification can also be used in which a tag with bindingaffinity for the subunits is fused, followed by affinity chromatographyusing a substance with binding affinity for this tag. Examples ofbinding tags that can be used include histidine tags comprising severalhistidines (His-Tag), β-D-galactosidase, glutathione S-transferase(GST), thioredoxin, maltose-binding protein, Myc, Xpress, and FLAG. IfGST is used, for example, the expressed protein can be purified easilywith a Glutathione Sepharose 4B column or such.

A substrate protein for measurement of kinase activity according to thepresent invention can be used as a substrate protein for use in themeasurement methods of the present invention, as previously described.In the present invention, a test compound is contacted with a substrateprotein and a Cdc7-ASK complex active substance in any of the ordersdescribed in the aforementioned i) to iii).

i) By contacting a test compound with a substrate protein, and thencontacting the Cdc7-ASK complex active substance, a kinase-modifyingeffect can be found in the action of the test compound on the substrateprotein. ii) By contacting a substrate protein with a Cdc7-ASK complexactive substance in the presence of a test compound, competitiveinhibitory activity on the kinase activity of the Cdc7-ASK complexactive substance can be evaluated. Furthermore, iii) By contacting asubstrate protein and Cdc7-ASK complex active substance, then contactinga test compound, the dephosphorylation effect of the test compound onthe phosphorylated substrate protein can be detected.

In the present invention, the level of phosphorylation of a substrateprotein can be measured according to methods similar to methods formeasuring the kinase activity of the present invention, as previouslydescribed. For example, the level of phosphorylation is measured by anantibody that recognizes the phosphorylated state at a phosphorylatedsite of a substrate protein.

The results of measuring the phosphorylation level are correlated with atest compound's effect on the kinase activity of the Cdc7-ASK complexactive substance. For example, when the phosphorylation level is reducedin comparison with a control that has not been contacted with a testcompound, the test compound is concluded to comprise the activity ofinhibiting phosphorylation. In addition, when the level ofphosphorylation increases due to a test compound, the test compound isconcluded to comprise the effect of promoting the kinase activity of aCdc7-ASK complex active substance.

Phosphorylation levels can be compared with a control that has not beencontacted with a test compound, as well as with the measurement resultsof a compound that clearly acts on kinase activity. For example, todiscover inhibitory effect on kinase activity, the level of a testcompound's inhibitory effect can be also evaluated by comparison with acompound already confirmed to comprise a certain degree of inhibitoryeffect. Furthermore, effects can be evaluated quantitatively byperforming an aforementioned measurement method on a substance whoseinhibitory effect on kinase activity has already been determined, andthen comparing these results with the measurement results of a testcompound.

Screening methods for compounds comprising the effect of regulatingCdc7-ASK complex kinase activity can be performed based on methods formeasuring the effect of a test compound on Cdc7-ASK complex kinaseactivity, according to the present invention. Namely, the presentinvention relates to methods of screening for compounds comprising theeffect of regulating Cdc7-ASK complex kinase activity, comprising thefollowing steps:

-   -   (a) measuring the effect of test compounds on Cdc7-ASK complex        kinase activity using an aforementioned method; and    -   (b) selecting test compounds that have a high or low level of        phosphorylation on comparison with a control.

In the screening methods of the present invention, phosphorylation levelcan not only be compared with a control that has not been contacted witha test compound, but also with the measurement results of a compoundclearly demonstrating an effect on kinase activity. For example, todiscover a kinase activity inhibitory effect, the level of a testcompound's inhibitory effect can also be evaluated by comparison with acompound already confirmed to comprise a certain degree of inhibitoryeffect. Compounds comprising an inhibitory effect above a constant levelcan then be screened by selecting compounds comprising a comparativelyhigher level of inhibitory effect.

In the screening methods of the present invention, examples ofsubstances that can be used as test compounds include natural orsynthetic proteins, peptides, antibodies, cell extracts from animals,plants or bacteria, culture supernatants, and low molecular weightcompounds. These test compounds can be obtained from compound librariesor gene libraries.

When the test compound is a natural ingredient, a single compound thatinhibits kinase activity can ultimately be identified by fractionatingeach test compound according to methods known to those skilled in theart (for example, various types of chromatography), and then detectingthose test compounds. Compounds inhibiting or promoting kinase activityand isolated by the screening can be used as agents for regulatingCdc7-ASK complex kinase activity.

Cdc7-ASK complex serves as a key cell growth factor in the living body.Thus, cell growth can be regulated by regulating Cdc7-ASK complex kinaseactivity. For example, compounds that inhibit Cdc7-ASK complex kinaseactivity are useful as cell growth inhibitors. More specifically,compounds comprising Cdc7-ASK complex inhibitory effect, and selectedaccording to the screening methods of the present invention, are usefulfor regulating cells such as cancer cells, whose growth should beinhibited.

The screening methods of the present invention specifically detectphosphorylation of a substrate protein by Cdc7-ASK complex. Thus, theeffect on Cdc7-ASK complex of compounds selected by the screeningmethods of the present invention can be said to be more specific. Whenusing such compounds to regulate cancer, for example, the resultingagents can be expected to be highly selective for proliferating cells,since they act specifically on cells in the growth phase.

These compounds are particularly useful as candidate compounds forcancer therapeutic agents. When using a compound isolated according tothe screening methods of the present invention as an agent to adjustkinase activity, that compound can be prepared for use in accordancewith known pharmaceutical production methods. For example, the compoundcan be administered to a patient along with a pharmaceuticallyacceptable carrier or medium (such as physiological saline, a vegetableoil, suspending agent, surfactant or stabilizer). Administration may beperformed transcutaneously, nasally, transtracheally, intramuscularly,intravenously, or orally, according to the properties of the compound.Although the dosage varies according to patient age and body weight,patient condition, administration method, and so on, those with ordinaryskill in the art can select a suitable dosage.

In addition, the present invention relates to a kit comprising anantibody capable of recognizing the phosphorylated state of a substrateused in the aforementioned measurement methods or screenings. When usedto detect kinase activity, kits of the present invention comprise, forexample, substrate proteins, buffers and so on, in addition to theaforementioned antibody. In addition, when used to screen for compoundsthat inhibit or promote kinase activity, Cdc7-ASK complex activesubstance is also included. Either the substrate protein or the antibodycan be labeled as previously described, while the other can beimmobilized.

An enzyme standard and substrate protein standard can be included in thekit in order to test the activity of the Cdc7-ASK complex activesubstance and the measuring system itself. Other ingredients can beadded to these standards and above-mentioned antibodies for the purposeof stabilization or such. For example, BSA at about 1%, or polyoles suchas sucrose and fructose at a final concentration of 0.2% to 10%(preferably 1%), can be added to the standards as protein denaturationpreventives after freeze-drying.

All prior art references cited herein are incorporated by reference inthe present specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the arrangement of motif-M and motif-C in human ASK,budding yeast Dbf4, and fission yeast Him1/Cfp1.

FIG. 2 shows results that confirm the specificity ofanti-Mcm2-phospho-S17 polyclonal antibody. Absorbance at 450 nm isplotted on the vertical axis, while antibody concentration (ng/ml) isplotted on the horizontal axis.

FIG. 3 shows the results of measuring the protein phosphorylationactivity of Cdc7-ASK complex active substance. Absorbance at 450 nm isplotted on the vertical axis, and the amount of enzyme (μL) is plottedon the horizontal axis.

FIG. 4 shows the results of investigating changes in reactivity when theconcentration of ATP added to a phosphorylation reaction solution waschanged, in a method for measuring Cdc7-ASK complex activity. Therelative activity value (%) based on a reactivity of 100 at 2 mM, isplotted on the vertical axis. The final ATP concentration (mM) in thereaction solution is plotted on the horizontal axis.

FIG. 5 shows the results of investigating changes in reactivity when thephosphorylation reaction time was changed, in a method for measuring theactivity of Cdc7-ASK complex. Absorbance at 450 nm is plotted on thevertical axis, while the reaction time (minutes) is plotted on thehorizontal axis.

FIG. 6 shows the results of evaluating a method for measuringphosphorylation activity by ELISA using Cdc7-ASK complex (wild type, WT)and inactive Cdc7-ASK complex (KD). Absorbance at 450 nm is plotted onthe vertical axis, while the amount of enzyme (μL) is plotted on thehorizontal axis.

FIG. 7 shows the results of measuring phosphorylation activity byradiofilter assay and ELISA method. The ELISA results (absorbance at 450nm) are plotted on the left vertical axis; the radiofilter assay results(radioactivity; cpm) are plotted on the right vertical axis; and theamount of enzyme (μL) is plotted on the horizontal axis.

FIG. 8 shows the results of measuring the effects of known proteinphosphorylation inhibitors on Cdc7-ASK complex phosphorylation activity.Relative inhibition value (%) is plotted on the vertical axis, whileinhibitor concentration (μM) is plotted on the horizontal axis.

BEST MODE FOR CARRYING OUT THE INVENTION

The following provide a more detailed explanation of the presentinvention based on Examples.

All commonly used techniques described in the Examples were carried outin accordance with the publications below. The methods introduced in thevarious publications can be used for the experimental methods used inthe Examples, without any particular restriction.

J. Sambrook, E. F., Fritsch & T. Maniatis (1989), “Molecular Cloning, alaboratory manual, second edition”, Cold Spring Harbor Laboratory Press;

Ed Harlow and David Lane (1988), “Antibodies, a laboratory manual”, ColdSpring Harbor Laboratory Press;

S. Oumi, K. Tsujimura & M. Inagaki (1994), “Cellular EngineeringSupplement, Experimental Protocol Series, Anti-peptide antibodyexperimental protocols”, Shujunsha Co., Ltd.; and

T. Migita, S. Konda, H. Motomoro & T. Hamaoka (1995), “ImmunologyExperimental Procedures I and II”, Nankodo Co., Ltd.

EXAMPLE 1 Identification of ASK Activity Domain

The present inventors conducted detailed mutation analyses using fissionyeast Hsk1-Him1/Dfp1 as a material. As a result, the present inventorsreported that the presence of only Dbf4-motif-m and Dbf4-motif-C issufficient for kinase activation (Ogino, K. et al., J. Biol. Chem. 276:31376-31387, 2001). Fission yeast Hsk1-Him1/Dfp1 is a complex thatcorresponds to human Cdc7-Dbf4. On the basis of this fact, the presentinventors predicted that a minimum domain comprising only motif-M andmotif-C would be also be adequate for kinase activation in human ASK.FIG. 1 shows the results of comparing the arrangement of motif-M andmotif-C in human ASK, budding yeast Dbf4, and fission yeast Him1/Cfp1.

More specifically, an amino acid sequence corresponding to the 173rd to349th residue (SEQ ID NO: 10) was selected from the amino acid sequenceof human ASK, shown in SEQ ID NO: 9. The DNA encoding this region isprecisely divided at introns. The region was selected because intronsare advocated to frequently exist at the divisions between functionaldomains or functional modules.

EXAMPLE 2 Vector for Expression of Cdc7-ASK Complex Active Substance

A plasmid designated as GST-TAT-ASK(minimum)-HA-huCdc7 was prepared inorder to express the active domains of human Cdc7 and ASK in E. coli.First, a GST-TAT-ASK-fused protein expression vector, GST-TAT11, wasprepared. A restriction enzyme site (NotI) was then inserted downstreamof ASK, and HA-Cdc7 was inserted at this site. At this time, a ribosomebinding sequence (RBS; Shine-Dalgano sequence) was added immediately infront of the HA-Cdc7. Due to the addition of this potent RBS, thesection from GST-TAT-ASK to HA-Cdc7 was transcribed as one sequence, andboth proteins could be efficiently translated from a singletranscription product. This detailed procedure is described below:

A region that encodes from the 173rd to the 349th amino acid of the cDNAof human ASK was amplified by PCR using the two primers below: MinimumASK-N (HindIII): CCC AAG CTT GAC ATT AGA TAC TAC (SEQ ID NO: 11) ATTGAA; and Minimum ASK-C (EcoRI): CCG GAA TTC TTT CTT TTT AGG TGT (SEQ IDNO: 12) GTC CTT.

The resulting amplification products were purified, and then cloned intoa GST-TAT11 vector. Each region is arranged in this vector in the orderof GST-TAT-HindIII-ASK-EcoRI. pGEX-2T-Tat11-TK vector was used as theGST-TAT11 vector. This vector comprises a structure in which a TATsequence comprising 11 amino acids (YGRKKRRQRRR/SEQ ID NO: 13) isinserted into a GST vector. The HindIII-EcoRI site of TK can be digestedto insert minimum ASK, which has been separately amplified with PCR.Since the HindIII frame is AAG CTT, translation in this vectorterminates immediately on reaching the stop codon in the 3FRAME, justdownstream of the EcoRI site.

Next, the EcoRI site of GST-TAT-HindIII-ASK-EcoRI was changed to a NotIsite using an EcoRI-NotI adapter (AATTGCGGCCGC/SEQ ID NO: 14). Thefull-length coding region was then amplified from the cDNA of humanCdc7, using the primers below: huCdc7-RBS-N (NotI)/: A TAA GAA TGC GGCCGC TAA Gaa gga (SEQ ID NO: 15) gAT ATA CAT atg TAC CCC TAC GAC GTG; andhuCdc7C-NotI: ATAAGAATGCGGCCGCTTATCACAAGCT (SEQ ID NO: 16) CATATCTTT.

In the nucleotide sequence of huCdc7-RBS-N(NotI), regions correspondingto RBS and ATG are indicated by lower case letters. The amino acidsequence encoded by the nucleotide sequence starting at ATG isMYPYDVPDYAFSPQRD (SEQ ID NO: 17). The MYPYDVPD of this sequencecorresponds to the HA epitope. With this vector, a protein is expressedthat comprises an amino acid sequence in which the amino acid sequencestarting at the 14th amino acid of human Cdc7 is coupled with the HAtag. As previously mentioned, both genes are translated due to theaddition of potent RBS.

Furthermore, wild type and kinase-deactivated type Cdc7 were prepared.The kinase-deactivated type is referred to as K9OE, which comprises anamino acid sequence in which the 90th amino acid, lysine (K), is mutatedto glutamic acid (E).

EXAMPLE 3 Expression and Purification of Cdc7-ASK Complex ActiveSubstance in Bacteria

E. coli C6001on- was transformed with the vector GST-TAT-ASK(minimum)-HA-huCdc7, constructed in Example 2 above, in accordance withordinary methods. C6001on- is a strain of E. coli that is missing 1on,which is a major E. coli protease.

The transformed E. coli was inoculated into 200 ml of LB mediumcontaining 40 μg/ml of ampicillin, and cultured at 37° C. When the OD⁶⁰⁰of the culture medium reached 0.5, IPTG was added at a concentration of1 mM, and then cultured for an additional three hours. The bacteria werecollected by centrifugation, washed, and then suspended in 20 ml ofbuffer A (40 mM Hepes/KOH (pH 7.6), 1 mM EDTA, 40 mM potassiumglutamate, 10% glycerol, and 1 mM DTT). The cells were disrupted usingultrasonic treatment, and the resulting products were fractionated intoa soluble fraction and a precipitate (insoluble fraction) bycentrifugation. 1 ml of glutathione Sepharose 4B was added to thesoluble fraction. After stirring at 4° C. for one hour, the glutathioneSepharose 4B was recovered by centrifugation, packed into a simplecolumn, and washed well with buffer A. The column was washed until theOD²⁸⁰ of the eluate reached 0.01 or less, using an amount of buffer Aequal to or more than 20 times the column volume. Subsequently, theglutathione Sepharose 4B was eluted first with buffer A containing 20 mMglutathione, and then with buffer A containing 50 mM glutathione.

The fraction finally eluted was separated by SDS-PAGE, and then silverstained, confirming the purification of GST-ASK (about 50 kDa) andHA-Cdc7 (about 65 kDa).

EXAMPLE 4 Expression and Purification of MCM2(1-130) by Bacteria

Total RNAs were prepared from Jurkat cells using ISOGEN (Nippon GeneCat. No. 311-02501), and cDNA was produced using You-Prime First-StrandBeads (Amersham-Pharmacia Cat. No. 27-9264-01). Using this cDNA as atemplate, human Mcm2(1-130) cDNA was then amplified using the ExpandHigh Fidelity PCR System (Boehringer-Mannheim Cat. No. 1732-650) and theprimers below. The resulting 390 bp PCR product was digested with theBamHI and XhoI restriction enzymes, and then cloned into the BamHI/XhoIsite of the E. coli expression vector, pGEX-4T-1 (Amersham-Pharmacia).The nucleotide sequence was determined, and the sequence of theresulting cDNA was confirmed to be human MCM2(1-130). The primers usedfor PCR are shown below: Forward: (SEQ ID NO: 18) 5′-CAC GGA TCC ATG GCATCC AGC CCG GCC CA-3′; and Reverse: (SEQ ID NO: 19) 5′-GTG CTC GAG CATCGC TGT CAT ACA GGA GCC-3′.

E. coli DH5a was transformed with MCM2(1-130)/pGEX-4T-1. This E. coliwas cultured for about five hours at 30° C., and the culturingtemperature was lowered to 20° C. when the optical absorbance (600 nm)reached 0.8. IPTG was added to a final concentration of 0.2 mM, andculture was continued for a further 16 hours at 20° C. The collected E.coli were suspended in ice-cold solubilizing buffer (PBS, 0.1%TritonX-100 and 1 mM PMSF) and dissolved by disrupting with anultrasonic crusher on ice. The dissolved crude extract was centrifugedfor 30 minutes at 15,000 rpm with a high-speed centrifuge to separatethe soluble and insoluble fractions. Each fraction was subjected toSDS-PAGE, and the GST-fusion Mcm2(1-130) was confirmed to be in thesoluble fraction. The soluble fraction was added to a column filled with2 ml of GSH-Sepharose 4B (Amersham-Pharmacia). The column was thenwashed with 50 ml of washing solution (20 mM Tris-HCl (pH 7.9), 0.5 MNaCl and 5 mM imidazole), and then eluate (10 mM glutathione and 50 mMTris-HCl (pH 9.6) was used to elute 2 ml into each of ten containers.Optical absorbance (A280 nm) was measured, and that with absorbance of0.1 or more was dialyzed overnight against ice-cold PBS. WesternBlotting using anti-MCM2-specific antibody confirmed that the dialyzedprotein was GST-fusion MCM2(1-130).

EXAMPLE 5 Identification of Phosphorylated Site of MCM2

First, the primary structures of human, mouse, frog, drosophila, fissionyeast, budding yeast, and so on were aligned to identify preservedserine and threonine residues. This was based on the hypothesis thatfunctionally important phosphorylation sites are preserved acrossspecies. Eight sites (F1, F2, F3, F4, F5, F6, NF1 and NF2) were selectedwhere these preserved serine and threonine residues formed clusters,being present in relatively large numbers.

Variant MCM2, in which serine and threonine residues 4 to 7 weresubstituted with alanine or glutamic acid at each site, was prepared foreach region. Variants were prepared according to methods that useoligonucleotides. They were then cloned into insect cell expressionvector, UW31. UW31 is a vector that can simultaneously express two typesof gene products. Recombinant virus solutions co-expressinghistidine-tagged MCM7 along with each variant MCM2 were obtained. Thesewere then co-infected into Sf9 insect cells together with a recombinantvirus solution that co-expresses histidine-tagged MCM4 and MCM6, andcell extracts were prepared. Affinity purification was then performed onthe MCM-2-4-6-7 complex using a nickel column.

The fractions into which the desired MCM-2-4-6-7 complex eluted werepooled. Next, the MCM-2-4-6-7 complex was further purified using amono-Q column (using the Pharmacia SMART System). The peak fractionseluting at 0.3 to 0.35 M NaCl were pooled. After dialyzing to a lowersalt concentration, the fraction was used as a substrate for in vitrophosphorylation reactions.

In addition, and at the same time, polypeptides comprising a portion ofthese preserved sites were prepared with a histidine or GST tag,expressed in E. coli, and then purified. They were prepared byamplifying the intended coding region using a primer oligonucleotide towhich a restriction enzyme site had been added, then cloning into ahistidine-tagged expression vector (pT7-7/pQE30) or GST-fusionexpression vector (pGEX-5X-3). The wild type and an alanine-substitutedvariant were expressed. These expression vectors were respectivelyinserted into strain DE3 or C6001on-, and their expression was induced.Affinity purification was then carried out with a nickel column orglutathione Sepharose bead column. These proteins were also used assubstrates for in vitro phosphorylation reactions.

When the substrate was MCM2-4-6-7 protein complex comprising variantMCM2, phosphorylation was not completely absent regardless of thevariant used. Therefore, the phosphorylated wild type and variant MCM2were digested with trypsin, and then spread out using two-dimensionalchromatography to analyze whether or not the spots had disappeared andthe phosphorylation sites were restricted.

In addition, as a result of phosphorylation by Cdc7, the SDS-PAGEmobility of MCM2, both in vivo and in vitro, shifts downward . Usingthis phenomenon as an indicator, an attempt was made to identifyvariants without the shift.

From these results, sites phosphorylated by the Cdk and Cdc7 of the MCM2N-terminal (S27/S41 and S26/S40) were identified. Moreover, thisphosphorylation was clearly a cause of the SDS-PAGE shift. The serineresidue at position 17 of human MCM2 (S17), which corresponds to theserine residue at position 26 of mouse MCM2 (S26), was demonstrated tobe efficiently phosphorylated by Cdc7 when a polypeptide of 130 aminoacids from the N-terminal was used as a substrate. On the basis of theseresults, a protein comprising 130 amino acids from the N-terminal ofhuman MCM2 (SEQ ID NO: 1) was used as a substrate protein for an ELISAassay, described below.

EXAMPLE 6 Production of Anti-Phosphorylated Peptide Antibody

Since it is clear that the Cdc7-ASK complex specifically phosphorylatesthe 17th serine residue of human Mcm2, an anti-phosphorylation-specificantibody that specifically recognizes the phosphorylated 17th serineresidue was next produced, as a tool for detecting phosphorylation ofthe 17th serine residue.

The following two peptides were produced using a peptide synthesizer,and correspond to the amino acid sequence from the 10th to the 20thresidue of the human MCM2 protein, comprising the 17th serine residuefound to be the phosphorylated site:

Phosphorylated peptide (written as Mcm2-phospho-S17): CRGNDPLTS(p)S (SEQID NO: 20); and

Non-phosphorylated peptide (written as Mcm2-S17): CRGNDPLTSS (SEQ ID NO:21).

The aforementioned peptide sequences are depicted using thesingle-letter code in the direction from the amino terminal to thecarboxyl terminal. “S(p)” indicates the phosphorylated serine residue.The cysteine (C) residue of the amino terminal was introduced to allowthe peptide to covalently bond with a carrier protein. Using HPLC, thesepeptides were confirmed to have a purity of 95% or more. Phosphorylatedpeptides were used as immunogens. Phosphorylated peptides andnon-phosphorylated peptides were used for measuring antibody titer andpurifying antibodies.

Alone, the synthesized short peptide has low antigenicity. Therefore,animals are generally immunized using peptides that have been bound tocarrier proteins. Albumin, myoglobin, hemocyanin and so on can be usedas carrier proteins, however, hemocyanin (keyhole limpet hemocyanin,hereinafter KLH; CALBIOCHEM) was used this time. The carrier protein wascovalently bonded using m-maleimidobenzoyl-N-hydroxysuccinimide ester(MBS; SIGMA), which is an agent that crosslinks phospho-Hs-S83 and KLH,in accordance with the attached protocol, and KLH-Mcm2-phospho-S17 wassynthesized.

20 μg/100 μl and 10 μg/100 μl of KLH-Mcm2-phospho-S17 were used torespectively immunize rabbits and mice once. 100 μl of Freund's completeadjuvant (Yatron) was then added. Immunogens were then prepared byemulsification. The rabbits were immunized by subcutaneous injection tothe back. Immunization was carried out from three to five times everytwo weeks. After blood was collected from the auricular vein, serum wasrecovered. Antibody titer was measured by ELISA using a microtiter plateon which Mcm2-phospho-S17 and Mcm2-S17 were immobilized.

Adequate antibody titer was confirmed, and from the next week, a cycleconsisting of blood collection (during the first week), rest (during thenext week) and immunization (during the final week) was repeated fourtimes. Blood was collected from the auricular vein in the same manner asfor titer confirmation. Approximately 60 to 70 ml of blood was collectedeach time. In the final blood collection, blood was collected directlyfrom the heart using a catheter.

The collected blood was allowed to stand overnight at 4° C., serum wasseparated from clot, and the serum in the supernatant was recovered.Ammonium sulfate at a final concentration of 50% was added to theseparated and recovered serum. The mixture was stired, and thenseparated by centrifugation. The minimum amount of PBS was added to theprecipitate containing the IgG fraction, completely dissolving it. Thesolution was then dialyzed against PBS. After completely equilibratingwith the PBS, the partially purified antibody fraction was applied to acolumn, and the antibody was purified.

Mcm2-phospho-S17 synthetic peptide was used to produce the specificationcolumn, while Mcm2-S17 synthetic peptide was used to produce theabsorption column. 1 mg of synthetic peptide was dissolved in 5 ml of0.1 M carbonate buffer, and then added to CNBr-activated Sepharose 4B(Pharmacia) equilibrated with 1 mM hydrochloric acid. This was gentlymixed overnight at 4° C., and added to a column. The column was washedwith 5- to 10-fold PBS, and equilibrated with 1 M Tris-HCl (pH 7.5), toblock the active groups remaining on the surface of the Sepharose 4B.After blocking, the column was washed and equilibrated with PBS, andused for antibody purification.

To purify the anti-phosphorylated peptide antibodies, the aforementionedpartially purified antibody fraction was passed through a specificationcolumn. After washing with PBS/0.1% TritonX-100, the anti-phosphorylatedpeptide antibodies adhered to the column were eluted with 0.17 Mglycine-HCl (pH 2.5). The eluted antibodies were immediately neutralizedby addition of a suitable amount of 1 M Tris-HCl (pH 8.0), and dialyzedagainst PBS to obtain anti-Mcm2-phospho-S17 antibody fraction. This wascompletely equilibrated with PBS, and then passed through the Mcm2-S17Sepharose 4B column, an absorption column. Passing through thisabsorption column removes antibodies that also react with thenon-phosphorylated peptides present in the anti-Mcm2-phospho-S17antibody fraction. Anti-phosphorylated peptide-specific antibodies passthrough the column without being adsorbed. The absorption column waswashed with PBS/0.1% TritonX-100. Antibodies bound to the absorptioncolumn were eluted with 0.17 M glycine-HCl (pH 2.5). Following elution,the-column was again equilibrated with PBS/0.1% TritonX-100, and theanti-Mcm2-phospho-S17 antibody fraction was repeatedly passed throughthe absorption column until the non-phosphorylated peptide antibodieswere almost completely absorbed. ELISA using a non-phosphorylatedpeptide-sensitized plate was used to confirm that the antibodies thatreact with non-phosphorylated peptides were removed. Specificity to thephosphorylated peptide was ultimately confirmed by ELISA using aphosphorylated peptide-sensitized plate.

The peptide was dissolved in 0.1 M carbonate buffer at a concentrationof 1 μg/ml. This was dispensed at 50 μl/well to each well of a 96-wellmicrotiter plate for ELISA. This was then left overnight at 4° C. tosensitize. Following sensitization, the peptide solution was removed, ablocking solution (1% BSA, 5% sucrose and 0.1% NaN₃/PBS) was added toeach.well at 200 μl/well, and the plate was allowed to stand for aboutone hour at room temperature. The blocking solution was then completelyremoved, followed by air-drying in a draft chamber (immobilization).

The plates on which phosphorylated peptide (Mcm2-phospho-S17) wereimmobilized were used to measure each antibody titer, confirm absorptionof anti-phosphorylated-serine-residue-specific antibody, and confirm thespecificity of the anti-phosphorylated-peptide antibody. In addition,plates on which non-phosphorylated peptide (Mcm2-S17) was immobilizedwere used to confirm column absorption of non-specific antibodies.

The serum for testing antibody titer was serially diluted. in 4-foldincrements, starting from a 200-fold dilution using PBS. Purifiedantibody was serially diluted in 4-fold increments starting from aconcentration of 1 μg/ml using PBS. The diluted samples were added tothe sensitized plates at 50 μl per well. After addition, the plates wereallowed to stand for one hour at room temperature (primary antibodyreaction). The reaction solution was then discarded and each well waswashed four or more times with PBS. Anti-rabbit immunoglobulin antibody(secondary antibody) labeled with horseradish peroxidase (HRP) was thensuitably diluted with PBS and added to each well in 50 μl aliquots. Thiswas then reacted for 30 to 60 minutes at room temperature (secondaryantibody reaction). Anti-rabbit IgG (H+L-chain) conjugated withperoxidase (MBL) was used as the secondary antibody. The secondaryantibody reaction solution was then discarded and each well was washedfour or more times with PBS. Coloring substrate solution (750 μMtetramethylbenzidine (TMB)) was then added to each well in 50 μlaliquots, and color was developed for five to 20 minutes at 30° C.(coloring reaction). The coloring reaction was stopped by adding 50 μlaliquots of reaction stopping solution (1.5 N H₃PO₄) to each well.Finally, optical absorbance at 450 nm was measured using a microplatereader.

Anti-Mcm2-phospho-S17 antibody was serially diluted in four-foldincrements from 4 μg/ml of antibody solution. Specificity was confirmedby ELISA using the plate on which phosphorylated peptide(Mcm2-phospho-S17) was immobilized, and the plate on whichnon-phosphorylated peptide (Mcm2-S17) was immobilized.

FIG. 2 shows the results of confirming the specificity ofanti-Mcm2-phospho-S17 polyclonal antibody. As a result, the opticalabsorbance of the plate on which Mcm2-phospho-S17 phosphorylated peptidewas immobilized increased with increasing antibody concentration. Theoptical absorbance of the plate on which Mcm2-S17 non-phosphorylatedpeptide was immobilized remained at nearly zero. Thus, the subjectantibody was proven to specifically recognize phosphorylation of the17th serine residue of human Mcm2.

EXAMPLE 7 Construction of an ELISA System for Measuring Cdc7-ASK ComplexPhosphorylation Activity

Regions comprising the 17th serine residue of human Mcm2, the substrateused, were selected in order to measure Cdc7-ASK complex phosphorylationactivity in a 96-well microtiter plate. More specifically, thesubstrates studied were 1) GST fusion proteins with the amino acidregions of residues 1 to 130 and 1 to 80 of human MCM2, whichphosphorylation assays using radioisotopes found to be a good substratefor Cdc7-ASK complex, and 2) a non-phosphorylated peptide correspondingto the amino acid sequence from the 10th to 20th amino acid residues(Mcm2-S17).

GST-Mcm2 (1-130 a.a.) and GST-Mcm2 (1-80 a.a.) were diluted to 5 μg/mlusing 0.1 M carbonate buffer, pipetted at 50 μl/well in to each well ofa microtiter plate for ELISA, and then sensitized overnight at 4° C. Thesensitizing solution was removed, a blocking solution (1% BSA, 5%sucrose and 0.1% NaN₃/PBS) was pipetted in to each well at 200 μl/well,and the plate was allowed to stand for about one hour at roomtemperature. The blocking solution was then completely removed. Theplates were then air-dried in a draft chamber, and stored at 4° C. untiluse. GST-Mcm2 (1-130 a.a.) and GST-Mcm2 (1-80 a.a.) were immobilized onthe inner walls of the microtiter plates by this procedure.

A protein phosphorylation reaction was then carried out in the wells inwhich recombinant proteins GST-Mcm2 (1-130 a.a.), GST-Mcm2 (1-80 a.a.)or non-phosphorylated peptide (Mcm2-S17) had been immobilized. ELISA wasthen carried out in succession in the same wells.

A recombinant Cdc7-ASK complex dilution series was prepared by dilutingfrom one-fold (×1) to 64-fold in two-fold increments using aphosphorylation buffer. 50 μl aliquots of this solution were added tothe wells of the immobilized plates (a protein phosphorylationreaction). After incubating at 30° C. for a fixed period of time, thephosphorylation reaction solution was discarded and each well was amplywashed four or more times with PBS. Primary antibody, that is,anti-Mcm2-phospho-S17 polyclonal antibody, was diluted with an antibodydiluent (1% BSA, 0.1% NaN₃/PBS) to a concentration of 1 μg/ml, added in50 μl aliquots to each well, and allowed to react for 30 to 60 minutesat room temperature (primary antibody reaction). The primary antibodyreaction solution was then discarded and each well was amply washed fouror more times with PBS. Anti-rabbit immunoglobulin antibody (secondaryantibody, MBL) labeled with horseradish peroxidase (HRP) was thendiluted 1000-fold with PBS and added to each well in 50 μl aliquots,then allowed to react for 30 to 60 minutes at room temperature(secondary antibody reaction). The secondary antibody reaction solutionwas then discarded and each well was washed four or more times with PBS.A coloring substrate solution was then added to each well in 50 μlaliquots, and color was developed for five to 20 minutes (coloringreaction). The coloring reaction was stopped by adding 50 μl aliquots ofreaction stopping solution to each well, and the optical absorbance at450 nm was measured using a microplate reader.

The results are shown in FIG. 3. These results clearly demonstrate thatthe protein phosphorylation activity of a Cdc7-ASK complex activesubstance can be measured by ELISA, using the anti-phosphorylatedpeptide antibodies provided in the present invention. Recombinantprotein GST-Mcm2 (1-130 a.a.) was determined to be the most preferablesubstrate for use in this phosphorylation activity measurement system.One reason why non-phosphorylated peptide (Mcm2-S17) cannot be used inthis activity measurement system is presumed to be difficulty inCdc7-ASK complex recognition of the target 17th serine residue, sincethe region comprising the phosphorylated site is short. Similarobservations also suggest the possibility that GST-Mcm2 (10-20 a.a.) canalso be provided as a substrate in the present measurement system.

FIG. 4 shows the results of investigating changes in reactivity when thefinal concentration of ATP added to the phosphorylation reactionsolution was changed from 1 μM to 2 mM, in the method for measuringCdc7-ASK complex activity using GST-Mcm2 (1-130 a.a.) andMcm2-phospho-S17 polyclonal antibody, as described above. Enzymeactivity is shown as a relative activity value (%), where activity at 2mM is taken as 100. Activity of 90% is indicated at 0.1 mM, and thereaction virtually plateaus at 1 mM. Although activity equal to or morethan 30% of the maximum activity value was indicated, even in thepresence of low concentrations of ATP, reactivity was clearly shown tobe ATP-dependent.

FIG. 5 shows the results of investigating changes in reactivity when thephosphorylation reaction time in the method. for measuring Cdc7-ASKcomplex activity was changed from 0 to 150 minutes. Increases inabsorbance were observed to be dependent on reaction time, and thereaction virtually plateaued at 90 minutes.

The use of other kinases in the present measurement system was examined.A GST-Mcm2 (1-130 a.a.)-sensitized plate and anti-Mcm2-phospho-S17polyclonal antibody were used. The aforementioned Cdc7-ASK complex (wildtype, WT) and inactive (KD) Cdc7-ASK complex were used as enzymes.Activity was measured for each dilution series.

The results are shown in FIG. 6. Optical absorbance, i.e.,phosphorylation of the 17th serine residue, was observed to decrease asWT Cdc7-ASK complex was diluted by up to four-fold. This was notobserved for KD Cdc7-ASK complex, in which phosphorylation activity isnot present. On the basis of these findings, the present measurementsystem was proved able to measure Cdc7-ASK complex-specificphosphorylation activity.

A radiofilter assay is a method for detecting substrate phosphorylationthat uses uptake of the radioisotope [γ-³²P]ATP into an acid-insolublefraction. It is frequently used in measuring phosphorylation activity.The sensitivity of this method and the ELISA measurement system providedin the present invention were compared.

A recombinant Cdc7-ASK complex dilution series diluted from one-fold(×1) to 64-fold in two-fold increments was prepared using aphosphorylation buffer in 1 μg of recombinant protein GST-Mcm2 (1-130a.a.), and then adjusted to a total volume of 50 μl. After incubating at30° C. for a fixed period of time, the reaction was stopped by adding 1ml of 10% trichloroacetic acid and 0.2% Na₄P₂O₇. The acid-insolublefraction was trapped in a GFC filter (Whatman), and washed three timesusing 2% trichloroacetic acid and 0.02% Na₄P₂O₇. The radioactivity ofthe [γ-³²P]ATP taken up by the substrate was counted with a liquidscintillation counter.

The results are shown in FIG. 7. The results are shown as relativevalues (%), where both the radioactive count (cpm) in the radiofilterassay, and the optical absorbance in ELISA, are taken to be 100% at theone-fold concentration of Cdc7-ASK complex enzyme. Compared to themeasurement system using radioisotope [γ-³²]ATP, the ELISA method couldmeasure about ⅛ the amount of enzyme, since the value was 100% up to ×8.In addition, while the amount of substrate used-in the ELISA method is acalculated value of 0.25 μg per well, the radiofilter assay requiresabout 1 μg, or four times this amount. Furthermore, in the ELISA methodthere was hardly any background interference in the absence of enzyme.Thus, these systems are more sensitive, require smaller amounts ofsubstrate and enzyme, and can be measured without using radioisotopes,demonstrating the superiority of the ELISA measurement systems providedby the present invention

K-252a (Calbiochem) and Staurosporine are known as kinase inhibitors.They are known as inhibitors of various kinases such as CaM kinase II,protein kinase A, protein kinase C, and protein kinase G. The ELISAsystems provided by the present invention were studied using K-252a andStaurosporine. Dilution series of each inhibitor were prepared, mixedwith Cdc7-ASK complex prepared with a phosphorylation assay buffer, andthen adjusted to a total volume of 50 μl. This was then added to wellsin which GST-Mcm2 (1-130 a.a.) was immobilized. Using an above-describedmethod, ELISA was then used to examine Cdc7-ASK complex phosphorylationactivity.

The results are shown in FIG. 8. FIG. 8 shows the relative inhibitionvalues (%), where the activity value without an inhibitor is taken as100. Since both K-252a and Staurosporine inhibitors are kinaseinhibitors with low specificity, inhibition of phosphorylation activityby Cdc7-ASK complex was concentration-dependent. The IC50 value was 2 μMfor both of these inhibitors. On the other hand, concentration-dependentinhibitory effect for Cdc7-ASK complex was not observed for Roscovitine,Olomoucine, and UO126. Roscovitine and Olomoucine are cdk2 inhibitors,and U0126 is a MAP kinase inhibitor. These kinase inhibitors were shownto lack Cdc7-ASK complex inhibition activity. Therefore, the ELISAsystem provided by the present invention was in fact shown to beeffective in screening for agents that inhibit Cdc7-ASK complexphosphorylation activity.

INDUSTRIAL APPLICABILITY

According to the present invention, the mechanism by which the substrateprotein, MCM2, is phosphorylated by Cdc7-ASK complex was elucidated. Onthe basis of this, substrate protein phosphorylation by the Cdc7-ASKcomplex could be more specifically evaluated. In other words, themethods for measuring phosphorylation effect of the present inventionare useful as methods in which the phosphorylating action of Cdc7-ASKcomplex can be specifically evaluated. For example, the kinase activityof Cdc7-ASK has been clearly shown to increase in actively growingcancer cells (Gene, 1998, April 28;211(1):133-40, “A human homolog ofthe yeast CDC7 gene is overexpressed in some tumors and transformed celllines.”, Hess G. F., Drong R. F., Weiland K. L., Slightom J. L.,Sclafani R. A., and Hollingsworth R. E., Cancer Research, Pharmacia,Upjohn, Inc., 301 Henrietta Street, Kalamazoo, Mich. 49001, USA). Thus,the growth ability of certain cancer cells can be predicted based on thekinase activity of Cdc7-ASK. In addition, by comparing the amount ofexpressed Cdc7 in numerous human cancer cells and normal cells, thepresent inventors also confirmed that Cdc7 is extremely stronglyexpressed in nearly all cultured cancer cells (including fibroblast celllines and lymphoid lines). Thus, Cdc7-ASK kinase activity, which can beevaluated by the methods of the present invention, is useful as acarcinogenesis marker. Cdc7-ASK kinase activity can be specificallyevaluated by the present invention. Thus, the growth ability of cancercells can be evaluated more specifically.

Moreover, by applying the aforementioned measurement methods, thepresent invention has realized methods for evaluating the effects oftest compounds on the phosphorylation function of the Cdc7-ASK complex,as well as screening methods based on these evaluation methods.

The present invention's methods for measuring Cdc7-ASK kinase activity,or of screening for compounds that regulate that activity, are usefulfor the. development of cancer medicaments and treatment methodstargeting Cdc7-ASK. Compared to using known intracellular kinases (suchas Cdk-Cyclin) or proteins as targets, the use of Cdc7-ASK as a targetoffers the following advantages:

Firstly, by targeting Cdc7-ASK, more reliable control of activity can beexpected. While Cdk-Cyclin is a family composed by numerous similargenes, Cdc7-ASK is composed of molecules whose structure is morelimited. Thus, by using Cdc7-ASK as a target, activity can be reliablycontrolled.

Secondly, using Cdc7-ASK as a target makes it possible to specificallycontrol cell growth. Cdc7-ASK is a factor required for the initiationand progress of the S phase. Upon loss of this activity the S phaseimmediately stops and cell growth is arrested. Moreover, the results ofexperiments using genetically manipulated mice suggest that interruptionof the S phase, namely DNA replication, can potentially cause abnormalstructures to accumulate in DNA, which when detected as DNA damage, caninduce p53 and eventually cell death. In other words, inhibition ofCdc7-ASK activity can effectively block progression of the S phase incancer cells, efficiently removing cancer cells by inducing cell death.

Thirdly, the use of Cdc7-ASK as a target is expected to lead to thediscovery of completely new cell growth inhibitors. The structure ofCdc7-ASK kinase is unique, even among members of the kinase family. Inaddition, the structure of ASK is also different from that of cyclinmolecules, on which considerable research has been conducted. Thisactivation is predicted to be due to a novel, and heretofore unknown,mechanism regarding kinase activation. Thus, searching for activityinhibitors that target Cdc7-ASK is highly likely to lead to thediscovery of completely new substances, undiscovered by the varioustypes of kinase screening to date. This means that, for example, newlead compounds may be discovered in the research and development ofanti-cancer agents.

Fourthly, induction of cell death can be expected when using Cdc7-ASK asa target. In response to the unexpected arrest of DNA replication, cellsactivate so-called check-point kinases, such as ATM, Chk1, and Cds1,which respond by delaying the progress of the cell cycle. If Cdc7-ASKinhibitors are used in combination with inhibitors against thesecheck-point kinases, enhanced induction of cell death, due to inhibitedS phase progression caused by inhibiting Cdc7-ASK activity, can beexpected.

In this manner, Cdc7-ASK complex plays important roles in the initialstages of cell growth in the cell cycle. Moreover, its phosphorylationactivity is proportional to the ability of cancer cells to grow. Inother words, cancer cell growth can be more specifically controlled bycontrolling Cdc7-ASK complex activity. Thus, the screening methods ofthe present invention are useful as methods for obtaining compounds thatcan specifically control cancer cell growth.

Moreover, the screening methods of the present invention are also ableto more specifically evaluate the effects of test compounds on thephosphorylation function of Cdc7-ASK complex. As a result, compoundsthat act more specifically on cancer can be selected.

In addition, the present invention has also provided a methods forpreparing the Cdc7-ASK complex active substances required by each of theaforementioned methods. According to the methods of the presentinvention, complexes comprising activity similar to that of the Cdc7-ASKcomplex can be easily prepared in large volumes by using prokaryoticcells.

Furthermore, the present invention provides substrate proteins that arephosphorylated by the Cdc7-ASK complex. The substrate proteins of thepresent invention are proteins that comprise the structure required forphosphorylation by the Cdc7-ASK complex (or Cdc7-ASK complex activesubstance). By using such substrate proteins the phosphorylation effectof Cdc7-ASK complex can be more specifically evaluated. The Cdc7-ASKcomplex active substances and substrate proteins prepared based on thepresent invention are useful in the aforementioned methods.

In addition, the present invention has provided antibodies that identifythe level of phosphorylation of substrate proteins phosphorylated by theCdc7-ASK complex or Cdc7-ASK complex active substance. The antibodies ofthe present invention identify the level of phosphorylation at specificsubstrate protein sites. These antibodies make it possible to morespecifically evaluate the phosphorylation effect of the Cdc7-ASK complexor Cdc7-ASK complex active substance. In addition, the use of theantibody of the present invention enables the phosphorylation effect ofthe Cdc7-ASK complex or Cdc7-ASK complex active substance to be easilyevaluated based on immunoassay principles.

1. A method for measuring the kinase activity of a Cdc7-ASK complex, comprising the following steps: (a) contacting a substrate protein with the Cdc7-ASK complex under conditions that allow phosphorylation of the substrate protein, wherein the substrate protein is a protein comprising the amino acid sequence of SEQ ID NO: 1, or a protein functionally equivalent to that protein; (b) measuring the level of phosphorylation of a serine residue of the substrate protein at the position corresponding to position 17 in the amino acid sequence of SEQ ID NO: 1; and (c) measuring the kinase activity of the Cdc7-ASK complex using the level of phosphorylation as an indicator.
 2. The method according to claim 1, wherein the level of phosphorylation is measured based on the level of binding of an antibody that identifies the level of phosphorylation of the serine residue.
 3. The method according to claim 1, wherein the Cdc7-ASK complex is derived from a biological sample.
 4. A method for measuring the effects of a test compound on the kinase activity of a Cdc7-ASK complex, comprising the following steps: (a) contacting a test compound, a substrate protein and a Cdc7-ASK complex active substance, wherein the substrate protein is a protein comprising the amino acid sequence of SEQ ID NO: 1, or a protein functionally equivalent to that protein, and wherein they are contacted in any of the following orders i) to iii): i) the test compound and substrate protein are contacted, followed by contacting the Cdc7-ASK complex active substance, ii) the substrate protein and Cdc7-ASK complex active substance are contacted in the presence of the test compound, or iii) the substrate protein and Cdc7-ASK complex active substance are contacted, followed by contacting the test compound; (b) measuring the level of phosphorylation for a serine residue of the substrate protein at the position corresponding to position 17 in the amino acid sequence shown in SEQ ID NO: 1; and (c) measuring the effect of the test compound on the kinase activity of the Cdc7-ASK complex active substance using the level of phosphorylation as an indicator.
 5. A method of screening for compounds comprising the effect of regulating the kinase activity of a Cdc7-ASK complex, comprising the following steps: (a) measuring the effect of a test compound on the kinase activity of the Cdc7-ASK complex according to the method described in claim 4; and (b) selecting a test compound with a high or low level of phosphorylation by comparison with a control that has not been contacted with the test compound.
 6. A screening method according to claim 5, wherein a compound having a low level of phosphorylation is selected in step (b) of claim
 5. 7. An inhibitor of cell growth comprising a compound selected according to the screening method of claim 6 as its active ingredient.
 8. A kit for measuring the kinase activity of a Cdc7-ASK complex comprising: (a) a substrate protein comprising a continuous amino acid sequence that comprises a serine residue at position 17 of the amino acid sequence of SEQ ID NO: 1, and that which is selected from the amino acid sequence of SEQ ID NO: 1; and (b) an antibody that identifies the level of phosphorylation of the serine residue of the substrate protein at the position corresponding to position 17 of the amino acid sequence of SEQ ID NO:
 1. 9. A kit for evaluating the effect of a test compound on the kinase activity of a Cdc7-ASK complex, comprising: (a) a Cdc7-ASK complex active substance; and, (b) a substrate protein comprising a continuous amino acid sequence that comprises the serine residue at position 17 of the amino acid sequence of SEQ ID NO: 1, and is selected from this amino acid sequence.
 10. A process for producing a Cdc7-ASK complex active substance comprising the following steps: (a) introducing a DNA encoding human Cdc7 protein and a DNA encoding a protein comprising the amino acid sequence described in SEQ ID NO: 10 or a protein functionally equivalent to the protein, into prokaryotic cells in a state that allows monocistronic expression; (b) expressing the two DNAs; and (c) recovering the expressed protein.
 11. An antibody that identifies the level of phosphorylation of the serine residue at position 17 of a protein comprising the amino acid sequence of SEQ ID NO:
 1. 12. A protein according to any of the following (a) to (d): (a) a protein comprising the amino acid sequence of SEQ ID NO: 1; (b) a protein comprising a continuous amino acid sequence that is selected from the amino acid sequence described in SEQ ID NO: 3, and comprises the serine of position 17; (c) a protein comprising an amino acid sequence in which one or more amino acids in the amino acid sequence of SEQ ID NO: 1 are substituted, deleted, added and/or inserted, wherein the protein is phosphorylated by human Cdc7-ASK complex; and (d) a protein comprising an amino acid sequence comprising 90% or more homology with the amino acid sequence of SEQ ID NO: 3, wherein the protein is phosphorylated by human Cdc7-ASK complex.
 13. A protein comprising a continuous amino acid that comprises the amino acid sequence of SEQ ID NO: 10 and that which is selected from the amino acid sequence of SEQ ID NO:
 9. 14. A polypeptide according to claim 13 that comprises the amino acid sequence of SEQ ID NO:
 10. 